c‐FOS is an integral component of the IKZF1 transactivator complex and mediates lenalidomide resistance in multiple myeloma

Abstract Background The immunomodulatory drug lenalidomide, which is now widely used for the treatment of multiple myeloma (MM), exerts pharmacological action through the ubiquitin‐dependent degradation of IKZF1 and subsequent down‐regulation of interferon regulatory factor 4 (IRF4), a critical factor for the survival of MM cells. IKZF1 acts principally as a tumour suppressor via transcriptional repression of oncogenes in normal lymphoid lineages. In contrast, IKZF1 activates IRF4 and other oncogenes in MM cells, suggesting the involvement of unknown co‐factors in switching the IKZF1 complex from a transcriptional repressor to an activator. The transactivating components of the IKZF1 complex might promote lenalidomide resistance by residing on regulatory regions of the IRF4 gene to maintain its transcription after IKZF1 degradation. Methods To identify unknown components of the IKZF1 complex, we analyzed the genome‐wide binding of IKZF1 in MM cells using chromatin immunoprecipitation‐sequencing (ChIP‐seq) and screened for the co‐occupancy of IKZF1 with other DNA‐binding factors on the myeloma genome using the ChIP‐Atlas platform. Results We found that c‐FOS, a member of the activator protein‐1 (AP‐1) family, is an integral component of the IKZF1 complex and is primarily responsible for the activator function of the complex in MM cells. The genome‐wide screening revealed the co‐occupancy of c‐FOS with IKZF1 on the regulatory regions of IKZF1‐target genes, including IRF4 and SLAMF7, in MM cells but not normal bone marrow progenitors, pre‐B cells or mature T‐lymphocytes. c‐FOS and IKZF1 bound to the same consensus sequence as the IKZF1 complex through direct protein‐protein interactions. The complex also includes c‐JUN and IKZF3 but not IRF4. Treatment of MM cells with short‐hairpin RNA against FOS or a selective AP‐1 inhibitor significantly enhanced the anti‐MM activity of lenalidomide in vitro and in two murine MM models. Furthermore, an AP‐1 inhibitor mitigated the lenalidomide resistance of MM cells. Conclusions C‐FOS determines lenalidomide sensitivity and mediates drug resistance in MM cells as a co‐factor of IKZF1 and thus, could be a novel therapeutic target for further improvement of the prognosis of MM patients.

but not IRF4. Treatment of MM cells with short-hairpin RNA against FOS or a selective AP-1 inhibitor significantly enhanced the anti-MM activity of lenalidomide in vitro and in two murine MM models. Furthermore, an AP-1 inhibitor mitigated the lenalidomide resistance of MM cells. Conclusions: C-FOS determines lenalidomide sensitivity and mediates drug resistance in MM cells as a co-factor of IKZF1 and thus, could be a novel therapeutic target for further improvement of the prognosis of MM patients.

K E Y W O R D S
activator protein-1, drug resistance, IKZF1 complex, immunomodulatory drugs, multiple myeloma

BACKGROUND
Multiple myeloma (MM) is a blood cancer characterized by the clonal proliferation of transformed plasma cells in the bone marrow. 1 The treatment outcome of MM patients has been greatly improved by the introduction of proteasome inhibitors as well as thalidomide and its analogues lenalidomide and pomalidomide, which are collectively designated immunomodulatory drugs (IMiDs). 2 More recently, therapeutic antibodies against CD38 and SLAMF7 have been incorporated into treatment strategies and quickly became an essential part of the current standard of care with IMiDs. 3 Among these key drugs, lenalidomide is arguably the most important and is continuously used during the course of MM treatment. 4 Low sensitivity to lenalidomide and the emergence of resistance lead to early treatment failure and late relapse during maintenance, respectively. 5 Therefore, the identification of the factors determining lenalidomide sensitivity is critical for further improvement of the treatment outcome of MM patients. IMiDs exert anti-myeloma action via direct binding to cereblon (CRBN), the substrate receptor of the CRL4 E3 ubiquitin ligase complex. 6 CRBN-bound IMiDs recruit IKZF1/Ikaros and IKZF3/Aiolos, members of the IKZF family of transcription factors, for ubiquitination and subsequent proteasome-dependent degradation. 7 The degradation of IKZF1 and IKZF3 results in the diminished expression of interferon regulatory factor 4 (IRF4), which is essential for the survival of MM cells, 8 and subsequent cell death. 6,7 Given the fundamental role of CRBN in the anti-myeloma activity of IMiDs, it is reasonable to speculate that its dysfunction underlies cell insensitivity and resistance to IMiDs. In fact, Gooding et al. 9 reported the association of genetic alterations of the CRBN gene, such as point mutations, structural abnormalities, and alternative splicing, with acquired resistance to both lenalidomide and pomalidomide in MM patients. However, the genetic changes in CRBN do not explain lenalidomide resistance in all cases, because the abnormalities, excluding exon 10 splicing, are detected in less than 10% of lenalidomiderefractory cases. 10,11 Therefore, it is still important to explore additional mechanisms of lenalidomide resistance in MM.
The IKZF family is widely expressed in the hematopoietic system and maintains homeostasis through the transcriptional regulation of a wide variety of genes involved in the proliferation and differentiation of blood cells. 12 The first member of the family, IKZF1/Ikaros, functions as a tumour suppressor in lymphoid lineages via transcriptional repression of NOTCH1 in association with the nucleosome remodelling and deacetylase (NuRD) complex and the Polycomb repressive complex 2. 13,14 In normal lymphoid cells, the repressor function of IKZF1 is dominant over its activator function for the maintenance of lineage fidelity. 15 In contrast, in MM cells, IKZF1 acts as a transcriptional activator to sustain the expression of IRF4 and other critical oncogenes. 16,17 It is tempting to speculate that IKZF1 forms a complex in MM cells that differs from the complex in normal lymphoid cells and that a component of the MM-specific IKZF1 complex mediates transactivator function. In support of this view, a recent report demonstrates that IKZF1 knockdown does not necessarily suppress the expression of IRF4 in MM cells. 18 This finding indicates the existence of an unknown factor that could work independently of and/or in cooperation with IKZF1 to mediate oncogenic transcription in MM cells and could promote IMiD resistance by maintaining IRF4 transcription after IKZF1 degradation.
In this study, we attempted to identify the factor that changes the IKZF1 complex from a transcriptional repressor to an activator in MM cells using bioinformatic approaches. We found that c-FOS, a member of the activator protein-1 (AP-1) family of transcription factors, 19,20 is an integral component of the IKZF1 complex and is primarily responsible for the activator function of the complex in MM cells. The expression levels of c-FOS appear to determine the sensitivity of MM cells to lenalidomide; c-FOS mediates lenalidomide resistance via a compensatory increase in the amounts of c-FOS bound on promoter regions of IKZF1-target genes after lenalidomide treatment. c-FOS may be a novel target for augmenting lenalidomide efficacy and overcoming lenalidomide resistance in MM cells.

Drugs
We purchased lenalidomide and T-5224 from Selleck Chemicals and other reagents from Sigma-Aldrich unless otherwise stated. Drugs were dissolved in dimethyl sulfoxide (DMSO) and used at a final dilution of 1/1000 to keep the final concentrations of DMSO < 0.1% to prevent alterations of drug effects or cell growth.

Construction and production of lentiviral expression vectors
We used the lentiviral short-hairpin RNA/shortinterfering RNA (shRNA/siRNA) expression vector pLL3.7 for c-FOS knockdown experiments. Oligonucleotides containing siRNA target sequences are shown in Table S1. Scrambled sequences were used as controls. These vectors were co-transfected into 293FT cells with packaging plasmids (Thermo Fisher Scientific) to produce infective lentiviruses in culture supernatants. Lentiviruses were then added to cell suspensions in the presence of 8 μg/ml polybrene and transduced for 24 h.

Quantitative real-time reverse transcription-PCR
Total cellular RNA was isolated using an RNeasy Kit (Qiagen), reverse-transcribed into complementary DNA using ReverTra Ace and oligo(dT) primers (Toyobo), and subjected to quantitative real-time reverse transcription-PCR using Expression Assays (Hs99999140 for FOS, Hs00180031 for IRF4, Hs00904275 for SLAMF7, Hs00936295 for BSG, Hs00152927 for CD48 and Hs01922876 for GAPDH) and TaqMan Fast Universal PCR Master Mix.

Chromatin immunoprecipitation assays
We used ChIP-IT Express Enzymatic Kit (Active Motif, Carlsbad, CA) to perform ChIP assays. 22 In brief, we fixed cells in 1% formaldehyde at room temperature for 10 min and isolated chromatin fractions using enzymatic shearing. After centrifugation, the supernatants were incubated with ChIP-grade antibodies against IKZF1/Ikaros (Active Motif 2A9) and c-FOS (Active Motif 2C5) or isotypematched controls in the presence of protein G magnetic beads at 4 • C overnight. We purified DNA fragments from the mixture and carried out quantitative PCR using Power SYBR Green Master Mix (Thermo Fisher Scientific) and the primers listed in Table S2. The fold enrichment of target genes between ChIP-DNA and input-DNA was determined by the 2 −∆∆Ct method. We outsourced the ChIP-seq analyses to Active Motif and deposited the data and protocols in the MINSEQE-compliant GEO database under accession numbers GSE194381 and GSE194387.

Protein-DNA binding assays
We examined whether IKZF1 and c-FOS formed a stable complex on DNA using an EpiQuik General Protein-DNA Binding Assay Kit (EpiGentek) as reported previously. 23 In brief, we incubated nuclear extracts with a biotinylated double-stranded oligonucleotide harbouring an IKZF-binding sequence, TCAGCTTTTGGGAATGTATTCCCTGTCA, 24 and captured the resultant IKZF-DNA complex on the bottom of streptavidin-coated plates. After vigorous washing, the residual complex was detected by sandwich immunoassay using an anti-IKZF1 antibody (Thermo Fisher Scientific 4E9) and a horseradish peroxidase-conjugated secondary antibody. The DNA-binding activity level was calculated according to the manufacturer's instructions. For antibody perturbation, we included specific antibodies against c-FOS (Cell Signaling Technology #2250) and IKZF1 (Cell Signaling Technology #14859) during the incubation of nuclear extracts with the oligonucleotide probe.

Murine MM models
Luciferase-expressing sublines of MM.1S cells were suspended in 100 μl of RPMI-1640 medium and subcutaneously inoculated into the right thigh of male nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice (Charles River Laboratories). Drugs were administered intraperitoneally in 200 μl of solution containing 5% DMSO (vol/vol) in sterile phosphatebuffered saline. The control group received only a vehicle following the same treatment schedule as the experimental group. The tumour burden was monitored by measuring tumour-derived luciferase activity with a noninvasive bioimaging system. In short, tumour-bearing mice were intraperitoneally injected with 1.5 mg of the luciferase substrate D-luciferin after being anaesthetized with isoflurane. Photons transmitted through the body were collected for a specified length of time and analyzed using the IVIS-CT Imaging System with Living Image software (Xenogen). The quantitative data are expressed as photon units (photons/s). [25][26][27] We also used an orthotopic MM model, in which luciferase-expressing KMS26 cells were engrafted into the bone marrow of NOD/SCID mice via tail-vein injection. All animal studies were approved by the Institutional Animal Ethics Committee and performed in accordance with the Guide for the Care and Use of Lab-oratory Animals formulated by the National Academy of Sciences.

Histopathological examination of inoculated tumours
Subcutaneous tumours were resected from euthanized mice, weighed, and embedded in the Tissue-Tek O.C.T. compound (Sakura Finetek) after formalin fixation. We prepared 5-μm-thick sections and subjected them to hematoxylin-eosin staining or immunohistochemical staining with mouse anti-CD138 (Thermo Fisher Scientific DL-101) and rabbit anti-IRF4 (Cell Signaling Technology #15106) antibodies as primary antibodies and Alexa Fluor 488-conjugated anti-rabbit IgG and Alexa Fluor 594-conjugated anti-mouse IgG (Thermo Fisher Scientific) as secondary antibodies. Nuclei were counterstained with DAPI (Cell Signaling Technology #8961). The stained samples were examined under a BZ-X fluorescence microscope (Keyence, Osaka, Japan).

Statistics
We used KaleidaGraph software (Synergy Software) to perform one-way ANOVA with the Student-Newman-Keuls multiple comparison test and Student's t-test to determine statistical significance. Kaplan-Meier survival curves were generated and analyzed by log-rank test using GraphPad Prism software (GraphPad Software). p-Values less than .05 were considered significant.

Co-binding of IKZF1 and c-FOS at transcription start sites of IKZF1-target genes in MM cells
To identify the unknown components of the MM-specific IKZF1 complex in situ, we comprehensively analyzed the binding of IKZF1 to the genome of the human MM cell line MM.1S using chromatin immunoprecipitation with high-throughput DNA sequencing (ChIP-seq). The genome-wide screening of IKZF1 binding detected 13,932 peaks, ∼60% of which were located in the vicinity of transcription start sites (TSSs) in MM.1S cells ( Figure 1). We confirmed the binding of IKZF1 at the TSSs of well-known targets, such as IRF4, SLAMF7 and BSG, 7,16,28,29 and identified novel candidate IKZF1-regulated genes, such as CNTRL, ISG20, OMA1, CD48, ICAM1, TIGIT and TET1, on the basis of enhancer/promoter binding ( Figure 2A). Next,   We determined the correlation of expression levels between IKZF1-target genes (y-axis) and the indicated genes (x-axis) using the GenomicScape tool (http://www.genomicscape.com). 32 The data were extracted from DNA microarray analyses of gene expression in newly-diagnosed MM patients. 53 (B) Cell lysates were prepared from the indicated MM cell lines and analyzed by immunoblotting using antibodies against c-FOS, we incorporated the results of ChIP-seq analyses of IKZF1 binding into the ChIP-Atlas platform 30,31 and investigated the co-occupancy of IKZF1 with other DNA-binding factors on the genome of MM cells. ChIP-Atlas analyses revealed that the AP-1 binding motif was present in nearly half of IKZF1-target genes and that c-FOS, a member of the AP-1 family, was actually co-localized with IKZF1 at histone H3K27-acetylated enhancer/promoter regions of these genes in MM cells ( Figure S1 and Figure 2B). Colocalization of IKZF1 and c-FOS was not evident on the IRF4 or SLAMF7 gene in normal bone marrow progenitors, pre-B cells or CD4-positive T-lymphocytes, although c-FOS alone bound to the IRF4 promoter in pre-B cells and IKZF1 alone bound to the SLAMF7 promoter in helper T-cells, probably reflecting their involvement in active transcription of respective genes in each cell type ( Figure  S2). To validate the co-occupancy of IKZF1 and c-FOS on the myeloma genome, we carried out ChIP-seq analyses of global c-FOS binding in MM.1S cells and superimposed the two images on the UCSC genome browser. The genomewide screening of c-FOS binding detected 10 173 peaks, ∼80% of which were located in the vicinity of TSSs in MM.1S cells (Figure 1). Whole-chromosome images generated by Strand NGS software revealed a significant overlap of the peaks representing IKZF1 and c-FOS binding to the myeloma genome ( Figure 1A, left panel, and Figure 2C). In detail, IKZF1 was co-occupied in 95% (9660/10 173) of c-FOS-binding sites, and conversely, c-FOS localization was observed in 69% (9660/13 932) of IKZF1-binding sites in MM cells ( Figure 1B, left panel). Binding motif analyses revealed that the consensus sequence 5′-ACTTCC(C/G)G-3′, which contains the GGAA core of the IKZF-binding motif in reverse orientation, 13 were significantly enriched and detected in 49.47% of IKZF1-binding sites and 46.58% of c-FOS-binding sites in MM.1S cells ( Figure 1C). This indicates that the two factors bind to the same sequence in approximately 50% of target genes. Co-occupancy was detected in canonical IKZF1 targets, such as IRF4 and SLAMF7, as well as in genes closely related to MM biology including CD38, MCL1, NSD2/MMSET, CCND1, PRDM1, IKZF1 and IKZF3 ( Figure 1B, right panel). Moreover, the two factors accumulated in the regions where histone H3K27 was heavily acetylated in genes expressed at high levels in MM cells ( Figure 3A and Figure S3A), whereas cooccupancy was not observed or was not co-localized with histone H3K27 acetylation in genes underexpressed in MM cells ( Figure 3B and Figure S3B). These results strongly suggest that c-FOS is a co-factor that works in concert with IKZF1 in the transcriptional activation of genes related to MM biology, in addition to IRF4.

c-FOS is actually involved in the transcriptional regulation of IKZF1-target genes in MM cells
Having demonstrated that c-FOS is bound to histone H3K27-acetylated regulatory regions of IKZF1-target genes, we investigated whether c-FOS is actually involved in the transactivation of these genes in MM cells. First, we analyzed the GenomicScape database of gene expression in primary MM cells from newly-diagnosed patients 32 and found a weak (R = 0.  The binding of the IKZF1 complex to oligonucleotides containing an IKZF consensus motif was measured by sandwich immunoassay and is shown as the relative activity against the data obtained in the absence of blocking antibodies. Antibody perturbation was carried out with isotype-matched immunoglobulin (Control), an anti-IKZF1 antibody, an anti-c-FOS antibody and a combination of the two antibodies. p < .05 by Student's t-test against Control (n = 5). (C) HEK293T cells were transfected with an empty vector (Mock) or expression vectors carrying HA-tagged full-length IKZF1 protein, the exon 1-exon 4 fragment, the exon 5-exon 6 fragment, or shorter than that of MM patients with lower expression of FOS when they were treated with Total Therapy 2/3 ( Figure 4). Next, we performed a shRNA-mediated knockdown of c-FOS to verify its role in the expression of IKZF1-target genes in MM cells. As anticipated, shRNA against FOS significantly reduced the expression of IKZF1-targets with co-binding of IKZF1 and c-FOS in enhancer/promoter regions, such as IRF4 and SLAMF7, but not the expression of IKZF1-targets lacking c-FOS co-occupancy, such as BSG and CD48 ( Figure 4D, left panel). The expression levels of FOS were positively and negatively correlated with the expression levels of IRF4 and BSG, respectively, in sh-FOS-treated cells ( Figure 4D, right panel). These results imply that c-FOS positively regulates the transcription of a subset of IKZF1-target genes in MM cells.

c-FOS is an integral component of the IKZF1 complex in MM cells
Co-occupancy of IKZF1 and c-FOS on the same consensus in ∼50% of target genes strongly suggests that the two factors form a complex on the myeloma genome. To determine whether IKZF1 and c-FOS interact physically or bind independently on the same regions, we performed immunoprecipitation-immunoblot assays using nuclear extracts from two MM cell lines, MM.1S and KMS12-BM. Immunoprecipitates with an IKZF1-specific antibody contained c-FOS and c-JUN in addition to IKZF3 but not IRF4 ( Figure 5A, left panel). Conversely, c-FOS immunoprecipitates contained IKZF1 and IKZF3 in addition to c-JUN but not IRF4 ( Figure 5A, right panel). Furthermore, an anti-c-FOS antibody perturbed the binding of the IKZF complex to the peptide containing a cognate IKZF-binding motif in a protein-DNA binding assay, suggesting that IKZF1/3 and c-FOS form a stable complex on the genome ( Figure 5B). Previous studies have shown that the physical interactions of IKZF1 isoforms are essential for the normal development of lymphoid cells. 33,34 The specification and homeostasis of the lymphatic system are profoundly affected by IKZF1 mutations that impair protein-protein interactions or alter the ratio of DNA-binding and non-DNA-binding isoforms. 15,34 To test which domains of the IKZF1 protein are involved in c-FOS binding, we generated HA-tagged fragments of different IKZF1 domains and expressed them in HEK293T cells together with FLAG-tagged c-FOS. c-FOS was found to be associated with the activation and dimerization domains, exons 5-6 and exon 7, respectively, of IKZF1 in immunoprecipitationimmunoblot assays using nuclear extracts from transfected cells ( Figure 5C). Consistent with this result, a structurebased prediction of protein-protein interactions using the AlphaFold2 program showed the binding of c-FOS peptides to the region sandwiched between exons 5-6 and exon 7 of IKZF1 ( Figure 5D). Forced expression of IKZF1 fragments with the ability of c-FOS binding induced the expression of IRF4, which is not usually transcribed in HEK293T cells ( Figure 5C). Collectively, our data suggest that c-FOS is an integral component of the IKZF1 complex in MM cells and primarily mediates activator functions of the complex via direct binding to IKZF1 on the myeloma genome.

c-FOS overexpression is associated with lenalidomide resistance in MM cells
Lenalidomide exerts anti-MM activity by inducing the degradation of IKZF1 and the subsequent repression of IRF4 transcription. 6,7 Given that c-FOS mediates the activator function of the IKZF1 complex in MM cells, it is possible that the expression levels of c-FOS modify the lenalidomide sensitivity of MM cells. To substantiate this hypothesis, we overexpressed c-FOS in MM.1S and KMS12-BM cells and found that forced expression of c-FOS significantly mitigated the cytotoxicity of lenalidomide at clinically relevant concentrations (< 4 μM) along with sustained expression of IRF4 ( Figure 6A). c-FOS overexpression did not considerably affect the baseline expression of IKZF1 in MM cells ( Figure S5) despite the existence of c-FOS-binding sites in enhancer regions of the IKZF1 gene. 35 The transcription factor-binding motif algorithm TRANSFAC detected 10 IKZF1-binding sites and a c-FOS binding site in the IRF4 promoter. Sites 4−9 were actually occupied by IKZF1 and c-FOS, and were transcriptionally active, as evidenced by histone H3K27 hyperacetylation in the ChIP-Atlas analyses ( Figure 6B, upper panel). Lenalidomide significantly decreased the amounts of IKZF1 on these sites to ∼20% in KMS12-BM cells; however, c-FOS binding to the corresponding sequences was increased in ChIP-quantitative PCR analyses ( Figure 6B, lower panel). An increase in c-FOS binding to IKZF-binding sites was also observed in the SLAMF7 the exon 7 fragment of IKZF1 together with a FLAG-tagged c-FOS expression vector. Nuclear extracts were isolated 24 h after transfection and immunoprecipitated with an anti-HA antibody, followed by immunoblotting with antibodies against HA tag (IKZF1), FLAG tag (c-FOS) or rabbit immunoglobulin (IgG). Red arrows denote the positions of the transfected IKZF1 fragments. (D) Structure-based prediction of IKZF1-c-FOS interactions using the AlphaFold2 program. promoter of lenalidomide-treated KMS12-BM cells ( Figure  S6). Because the viability of KMS12-BM cells was reduced to only 70% with 2.5 μM lenalidomide ( Figure 6A, right panel), the discrepancy between the cytotoxic effects and IKZF1 down-regulation might be due to increased binding of c-FOS to the IKZF1 consensus and subsequent enhancement of the activity of the residual IKZF1 complex to maintain IRF4 transcription. In support of this hypothesis, IRF4 expression was decreased only to ∼80% at mRNA and protein levels by lenalidomide treatment in these experiments ( Figure 6C). To corroborate the role of c-FOS in lenalidomide resistance in a physiological context, we established lenalidomide-resistant sublines of NCI-H929 cells via continuous exposure of parental cells to the drug. As expected, c-FOS expression was readily up-regulated in lenalidomide-resistant sublines compared with the parental cell line along with the increased expression of IRF4 ( Figure S7A). Overall, c-FOS might be implicated in lenalidomide resistance and could be a therapeutic target for overcoming the resistance to and/or enhancing the anti-MM action of lenalidomide.

Genetic and pharmacological inhibition of c-FOS enhances the anti-myeloma activity of lenalidomide in vitro
To obtain proof of the above concept, we investigated whether disruption of the physical and/or functional interactions of IKZF1 with c-FOS increases the sensitivity of MM cells to lenalidomide. First, we transduced MM cell lines with shRNA against FOS and confirmed a > 60% reduction in the expression of FOS mRNA and c-FOS protein in lenalidomide-treated MM cells ( Figure  S8A,B). The shRNA-mediated c-FOS down-regulation significantly exacerbated the lenalidomide-induced decrease in the expression levels of IRF4 transcripts as well as diminished cell viability in MM.1S and KMS12-BM cells ( Figure 6D and Figure S8C). Next, we carried out the same experiments using the selective AP-1 inhibitor T-5224 (3-{5-[4-(cyclopentyloxy)−2-hydroxybenzoyl]−2-[(3hydroxy-1,2-benzisoxazol-6-yl)methoxy]phenyl} propionic acid). This compound was reported to inhibit AP-1 activity via interference with the physical interaction of c-FOS and DNA without altering the abundance of c-FOS/c-JUN [36][37][38] ; however, T-5224 modestly decreased the expression levels of c-FOS in our experiments ( Figure S7B). Treatment of MM cells with T-5224 alone did not affect the expression of IRF4 or cell viability but significantly enhanced the cytotoxicity of lenalidomide in both cell lines ( Figure 6E). Furthermore, lenalidomide and T-5224 showed additive/synergistic effects in isobologram analysis of drug combinations in MM.1S cells and at least additive effects in KMS12-BM cells ( Figure S9). The combined effects were less prominent in KMS12-BM cells treated with higher doses of lenalidomide because of the concave pattern of the drug response ( Figure 6A, right panel). In addition, we confirmed the combined cytotoxic effects of lenalidomide and T-5224 in patient-derived MM cells ( Figure S10). We also evaluated whether lenalidomide resistance is attenuated by the combination with T-5224 in NCI-H929LR cells. As expected, lenalidomide alone did not reduce the expression level of IRF4 or cell viability at all in lenalidomide-resistant NCI-H929 sublines, but the addition of T-5224 apparently overcame lenalidomide resistance as indicated by decreased IRF4 expression and cell viability ( Figure S7B,C).

A selective AP-1 inhibitor enhances the anti-myeloma activity of lenalidomide in vivo
Finally, we attempted to replicate the combined effects of lenalidomide and T-5224 in a murine MM model, in which luciferase-expressing sublines of MM.1S were subcutaneously inoculated into NOD/SCID mice. 25  measurable tumours developed, mice were randomly assigned to four groups (n = 5 per group) and treated with vehicle alone (0.9% NaCl), T-5224 alone, lenalidomide alone, or lenalidomide plus T-5224. The combination of lenalidomide and T-5224 significantly slowed the growth of inoculated tumours, as evidenced by the decrease in ex vivo luciferase activity ( Figure 7A and Figure S11) and in the sizes of the tumours resected on day 25 ( Figure 7B), whereas T-5224 or lenalidomide alone did not affect the growth of MM cells at the dose and schedule used in this experiment. Immunofluorescent staining revealed a marked decrease in cell numbers and IRF4 expression in inoculated tumours from the combined treatment group but not in those from other groups ( Figure 7C). We also investigated the efficacy of the two drugs in an orthotopic MM model, in which luciferase-expressing KMS26 cells were transplanted into NOD/SCID mice via tail-vein injection and engrafted in bone marrow after 14 days on average. After confirming engraftment by measuring ex vivo luciferase activity, we divided recipients into four groups (n = 5), in which the sum of luciferase activity was not statistically different (vehicle-treated group; 3.58 ± 1.34, lenalidomide-treated group; 4.04 ± 0.37, T-5224-treated group; 5.09 ± 0.78, lenalidomide and T-5224treated group; 4.03 ± 0.47 × 10 4 photons/s, P = 0.2327 by one-way ANOVA), and started the treatments with vehicle alone, T-5224 alone, lenalidomide alone, or the combination of lenalidomide and T-5224. As shown in Figure 7D, treatment with lenalidomide or T-5224 failed to prolong the survival of KMS26-transplanted mice; however, the combination of the two agents led to a significant increase in overall survival compared with the single-agent therapies. No obvious toxicities were observed in any group of mice (data not shown).

DISCUSSION
Lenalidomide exerts pharmacological action through the ubiquitin-dependent degradation of IKZF1 and subse-quent down-regulation of IRF4, a critical factor for the survival of MM cells. 6-8 IKZF1 functions as a tumour suppressor via transcriptional repression of oncogenes in normal hematopoietic lineages, [13][14][15] whereas it activates IRF4 and other oncogenes in MM cells. [16][17][18] These results suggest that the IKZF1 complex switches its function from transcriptional repressor to activator in MM cells; however, little is known about the underlying mechanisms of this switch. In this study, using bioinformatic and biological approaches, we determined that c-FOS, a bona fide transcriptional activator of the AP-1 family, is an integral component of the IKZF1 complex and is primarily responsible for the activator function of the complex in MM cells.
Because the binding sequence of c-FOS on the myeloma genome is different from common AP-1-binding motifs, such as TPA-responsive elements and cAMP-responsive elements, 19,20 it is highly possible that c-FOS binds to DNA through protein-protein interactions with IKZF1. This is the first report of the complex formation of IKZF1 and c-FOS as well as the disease-specific functions of this complex ( Figure 8, left panel). The MM-specific IKZF complex is composed of IKZF1/IKZF3 and c-FOS/c-JUN but does not contain IRF4, in contrast to a previous finding that IRF4 is a component of the IKZF complex in normal plasma cells. 39 The IKZF-IRF4 complex is associated with the NuRD complex to mediate repressor functions during normal plasma cell differentiation. 39 The exclusion of IRF4 might be involved in the switch of the IKZF complex from a repressor to a transcriptional activator during plasma cell transformation. AP-1 is a transcription factor complex composed of subunit proteins from the FOS (c-FOS, FOSB, FRA-1 and FRA-2), JUN (c-JUN, JUNB and JUND), ATF and MAF subfamilies, with a c-FOS/c-JUN heterodimer as a representative complex. 19,20 Each subunit carries basic leucine zipper domains, which promote dimerization via hydrophobic stretches and interactions with DNA via positively charged amino acids. 19 AP-1 regulates the proliferation, differentiation, and cell death of various cell types, including MM cells. 20  Tumor sections were prepared from mice and stained with hematoxylin and eosin (HE). Lower panel: Serial tumour sections were immunostained for IRF4 (green) and CD138 (red), followed by counterstaining with DAPI (blue). Merged images are shown at the bottom. The data are representative of several independent experiments. (D) We transplanted luciferase-expressing KMS26 cells (1 × 10 6 cells/mouse) into BALB/c mice via tail-vein injection. After confirming the engraftment of transplanted cells on day 14 via ex vivo luciferase assays, we assigned recipients to four groups (n = 5), which were standardized on the basis of total luciferase activity, and started the treatments with vehicle alone (0.9% NaCl, three times a week), 20 mg/kg T-5224 (three times a week), 10 mg/kg lenalidomide (three times a week), or the combination of T-5224 and lenalidomide (20 mg/kg T-5224 and 10 mg/kg lenalidomide, three times a week) intraperitoneally for 3 weeks. p-Values and hazard ratios (95% confidential interval) were calculated by log-rank test between the combined treatment group (red) and the lenalidomide-alone (green) or T-5224-alone treatment group (blue). No significant differences were observed between the vehicle-treated group (Control) and the lenalidomide-alone or T-5224 alone-treatment group. reported that c-FOS/c-JUN activity is altered in almost all MM patients and plays a major role in a transcriptional network associated with myeloma survival. 40 Fan et al. demonstrated that JUNB facilitates the proliferation, survival, and drug resistance of MM cells in the bone marrow microenvironment. 41,42 Furthermore, Ohguchi et al. identified c-FOS as a crucial factor for MM cell growth and survival downstream of the histone demethylase 6B-MAP kinase pathways. 43 AP-1-binding motifs are present in enhancers of several genes whose activity is increased in MM cells, and c-FOS binding was actually detected at the MAF gene promoter. 44,45 Consistent with the unique role of AP-1 in MM biology, Liu et al. identified AP-1-overexpressing subpopulations in MM patients using single-cell genomics. 46 Although the drug sensitivity of AP-1 high subclones was not described in their study, this subpopulation seems to possess malignant characteristics because they appear during disease progression or relapse. 46 Given the principal role of c-FOS in the activator function of the IKZF1 complex in MM cells, the sole targeting of IKZF1/IKZF3 by lenalidomide might be insufficient for inducing the complete down-regulation of IRF4 expression and subsequent cell death because residual AP-1 activity could sustain the transcription of IRF4 (Figure 8, middle panel). Similarly, an increase in c-FOS expression was observed in lenalidomide-treated non-small-cell lung cancer cells and is speculated to be a mechanism underlying the relative insensitivity of lung cancer to lenalidomide. 47 Considering these findings, the addition of AP-1 inhibitors may be a strategy for overcoming lenalidomide resistance and/or augmenting the efficacy of lenalidomide.
In this research, we demonstrated that a selective AP-1 inhibitor, T-5224, significantly enhanced the anti-MM activity of lenalidomide in vitro and in two murine MM models (Figure 8, right panel). Among AP-1 inhibitors, T-5224 is a lead compound that specifically inhibits the binding of AP-1 to regulatory regions of target genes, such as inflammation-related genes, 36,37 and is currently under phase II clinical trials for patients with rheumatoid arthritis and other inflammatory diseases after the absence of serious side effects was confirmed in phase I trials (JapicCTI-101359). 38 The clinical relevance of AP-1 inhibitors is supported by a recent report that T-5224 was able to restore the sensitivity of acute myeloid leukaemia cells to cytosine arabinoside. 48 The spectrum of clinical applications of AP-1 inhibitors might be expanded to MM in combination with lenalidomide in the future.
Finally, it should be noted that c-FOS may be involved in high-risk features of MM. For example, c-FOS is known to be positively regulated by the histone H3K36 methyltransferase MMSET/NSD2, which is ectopically expressed due to the chromosome translocation t(4;14) and might contribute to the overexpression of IRF4 and SLAMF7 as a direct transactivator in high-risk MM with t(4;14). 49,50 Lenalidomide resistance is a well-characterized clinical feature of MM patients with t(4;14) 51,52 and might be mediated via c-FOS overexpression under the influence of MMSET. According to Annunziata et al., the shRNA-mediated knockdown of MMSET reduces c-FOS expression in t(4;14)-harbouring MM cells. 45 These results suggest that c-FOS may be an important therapeutic target in high-risk MM patients, who are usually highly resistant to lenalidomide. 4,5

CONCLUSIONS
In conclusion, c-FOS determines lenalidomide sensitivity and mediates drug resistance in MM cells as a co-factor of IKZF1 and thus, could be a novel therapeutic target for further improvement of the prognosis of MM patients.

A C K N O W L E D G E M E N T S
The authors are grateful to Ms. Mayuka Shiino, Ms. Mai Tadaki and Ms. Akiko Yonekura for technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research from JSPS (to Naoki Osada, Jiro Kikuchi and Yusuke Furukawa) and research grants from the International Myeloma Foundation Japan (Jiro Kikuchi) and Kanehara Foundation (Naoki Osada). Naoki Osada received the 2021 Young Investigator Award from Jichi Medical University and the Japanese Society of Myeloma Research Award.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The ChIP-seq data were deposited in the MINSEQEcompliant GEO database under accession numbers GSE194381 and GSE194387.